Fitc conjugated secondary antibody

Cells were washed with cold‐PBS three times, fixed with 4% formaldehyde for 60 minutes and permeabilized using 0.1% triton‐x for 30 minutes. After blocking with 10% normal goat serum for 1 hour at room temperature, the samples were incubated with primary antibodies (anti‐VEGF‐A; 1:1000; Cell Signaling Technology, Massachusetts, USA) for 1 hour at room temperature and gently washed three times in PBS. Then, the samples were incubated with fluorescein (FITC)‐conjugated secondary antibody (1:1000; Cell Signaling Technology) and stained with DAPI (1 μg/mL, Cell Signaling Technology) before image acquisition using a fluorescence microscope (Olympus, Tokyo, Japan).

The two assays were utilized to localize LINC01134 and IGF2BP2 in SNU-182 and SK-HEP-1 cells. At first, 4% PFA was utilized for 15 min fixation at 37°C. After being permeabilized with 0.5% Triton X-100 (R00285, Leagene, Beijing, China), HCC cells were co-cultured with LINC01134 FISH probe in hybridization buffer and then stained with DAPI.
For the IF assay, IGF2BP2 primary antibody was added to incubate with cells at 4°C for a whole night. Then, FITC-conjugated secondary antibody (7076, Cell Signaling Technology, Boston, MA, United States) was added. With the help of a confocal laser microscope (Axio-Imager_LSM-800, Zeiss, Oberkochen, Germany), images were gained. The experiment was independently conducted in triplicate.

BC cells seeded on coverslips were subjected to incubation overnight at 4℃ with the corresponding specific antibody (TAF15, 1:200, Abcam) and with FITC-conjugated secondary antibody (1:200, Cell Signaling Technology) for 1 h at 37℃. Subsequently, the cells were cultivated for 13 h at 45℃ using a probe (5’CY3- AGAAGAGGCCTTGGGATTGGTTCGCTGC—3’CY3) (Geneseed) and then counterstained with DAPI.

Treated cells were harvested using trypsin and seeded on slides the day before immunofluorescence assays. The next day, cells were fixed with 4% formaldehyde, followed by the treatment with 1% Triton X-100 (Sigma, St. Louis, MO, USA) for permeabilization and then blocked with 2% bovine serum albumin for 40 min at room temperature. To visualize p-CREB, coverslips were incubated with an anti-p-CREB antibody overnight and then incubated with a FITC-conjugated secondary antibody (Cell Signaling Technology) for 1 h. DAPI (Sigma) was included during staining to visualize the nuclei of cells. Images were captured using a LSM880 + Airyscan confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 20X objective (NA 0.8). The fluorescence intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) based on previously described procedures^{21 (link)}.

The atrium tissue samples were fixed and sectioned for analysis as previously described.³¹ The atrium tissue sections were incubated with anti‐PU.1 (1:100; Abcam), anti‐PCNA (5 μg/mL; Abcam) and anti‐α‐SMA (1:50; Cell Signaling Technology) primary antibodies at 4℃ overnight, followed by incubation with a fluorescein isothiocyanate (FITC)–conjugated secondary antibody (1:1000; Cell Signaling Technology) and visualization with an Olympus BX51 microscope (Olympus Corporation).
For immunofluorescence assays of the atrial fibroblasts, the cells were washed with phosphate‐buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 min, followed by permeabilization with 0.1% Triton X‐100 in PBS. The cells were then added to coverslips with a syringe, and the coverslips were blocked with 3% bovine serum albumin for 30 min and then incubated overnight with primary antibodies against PU.1 (1:100; Abcam), anti‐PCNA (5 μg/mL; Abcam) and anti‐α‐SMA (1:50; Cell Signaling Technology). The next day, the coverslips were incubated with a FITC‐conjugated secondary antibody for 1 h, and the nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) for 30 min. Cells were then imaged with an Olympus BX51 microscope.

Antibodies for Nrf2, phosphorylated IKK, IKK-α, IKK-β, phosphorylated IκB, p65, phosphorylated JNK, total JNK, phosphorylated p38, total p38, phosphorylated ERK1/2, total ERK1/2, HO-1, an FITC-conjugated secondary antibody, and U0126 (ERK1/2 inhibitor) were purchased from Cell Signaling Technology (Beverley, MA, USA). A GCLM antibody and SB202190 (p38 inhibitor) were ordered from Abcam (Cambridge, UK), an iNOS antibody was supplied by BD Biosciences (Franklin Lakes, CA, USA), an actin antibody was purchased from Millipore (Bedford, MA, USA), and horseradish peroxidase–conjugated secondary antibodies were provided by Santa Cruz Biotechnologies (Santa Cruz, CA, USA). A protease inhibitor cocktail, a phosphatase inhibitor cocktail, and SP600125 (a JNK inhibitor) were supplied by Calbiochem (Merck Millipore, Darmstadt, Germany). Cell Culture Lysis Reagent was purchased from Promega (Madison, WI, USA). Bradford reagent was purchased from Bio-Rad (Hercules, CA, USA). Fetal bovine serum was acquired from Invitrogen (Carlsbad, CA, USA). Cell culture media, dimethylsulfoxide (DMSO), and other chemicals were ordered from Sigma Chemical Company (St. Louis, MO, USA).